The extracellular matrix (ECM) has been viewed as a static three dimensional scaffold that supports cells and tissues. However, our recent molecular imaging studies in living osteoblasts have shown that the ECM is highly dynamic and that ECM molecules form structures that continually undergo movement and deformation, mediated by cell-generated mechanical forces. These studies also suggest a novel role for cell movement in ECM assembly and reorganization. Fibronectin is one of the earliest proteins to be assembled into the ECM and facilitates assembly of other matrix proteins. In the previous funding cycle, using fibronectin null cell culture models and targeted gene deletion in osteoblasts, it was shown that fibronectin is essential for assembly of multiple bone ECM components, including type I collagen, fibrillin-1, Latent TGF2 binding protein-1, decorin and biglycan and is also required for normal mineralization. Fibronectin depletion also inhibits osteoblast differentiation. Fibronectin's effects on differentiation can be rescued by supplementation with BMP2, whereas its effects on ECM assembly and mineralization cannot, suggesting that fibronectin may regulate osteoblast differentiation via ECM targeting of osteogenic growth factors. Based on these observations, the proposed studies are centered on two main hypotheses. The first is that fibronectin is a multifunctional regulator of osteoblast differentiation and function through its effects as a central orchestrator for assembly of bone ECM proteins and through its role in ECM regulation of growth factor activity. The second is that dynamic cell movement is essential for the assembly and reorganization of bone ECM proteins. To test these hypotheses, in vitro and in vivo approaches will be used in combination with live cell imaging. Aim 1 will define the cascade of assembly of bone ECM proteins and its integration with cell and matrix dynamics. This will be done using fibronectin null osteoblast models in conjunction with live cell molecular imaging of bone ECM proteins and quantification of cell and fibril dynamics by computational analysis. Aim 2 will further define the role of fibronectin in osteoblast differentiation through regulation of BMP signaling. Live cell imaging techniques will also be used with osteoblast/osteocyte lineage reporters and fluorescent probes for ECM components to determine how osteoblast differentiation and BMP signaling is dynamically integrated with ECM assembly and reorganization. Aim 3 will use novel imaging probes to determine the dynamics of collagen assembly into the ECM of osteoblasts and the role of fibronectin in collagen deposition in vitro and in vivo. The studies will provide fundamental insights, from a dynamic perspective, into the mechanisms of assembly of bone ECM and how the ECM regulates osteoblast function. The data generated will significantly advance our understanding of the molecular and dynamic mechanisms underlying bone formation and have key implications for skeletal diseases such as osteoporosis, arthritis, osteogenesis imperfecta and bone diseases related to ECM proteins.